Fernando J. Díaz-Benjumea

Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila

The adult appendages of Drosophila develop from imaginal discs. An early step in disc patterning involves the formation of developmental boundaries that subdivide the discs into compartments. Anterior and posterior compartments are established in the embryo. Later in development a new boundary originates to subdivide the wing disc into dorsal and ventral compartments, which correspond to the dorsal and ventral surfaces of the adult wing. We report here that spatially localized expression of the homeobox gene apterous (ap) specifies the identity of dorsal cells in the wing. The boundary of cell lineage restriction between dorsal and ventral compartments coincides with the limit of the domain of ap expression. Using genetic mosaics, we show that juxtaposition of dorsal and ventral cells induces formation of the wing margin. We present evidence that the dorsal-ventral boundary promotes growth and serves as a pattern-organizing center in the wing disc.

Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development.

Nab proteins form an evolutionarily conserved family of transcriptional co-regulators implicated in multiple developmental events in various organisms. They lack DNA-binding domains and act by associating with other transcription factors, but their precise roles in development are not known. Here we analyze the role of nab in Drosophila development. By employing genetic approaches we found that nab is required for proximodistal patterning of the wing imaginal disc and also for determining specific neuronal fates in the embryonic CNS. We identified two partners of Nab: the zinc-finger transcription factors Rotund and Squeeze. Nab is co-expressed with squeeze in a subset of neurons in the embryonic ventral nerve cord and with rotund in a circular domain of the distal-most area of the wing disc. Our results indicate that Nab is a co-activator of Squeeze and is required to limit the number of neurons that express the LIM-homeodomain gene apterous and to specify Tv neuronal fate. Conversely, Nab is a co-repressor of Rotund in wing development and is required to limit the expression of wingless (wg) in the wing hinge, where wg plays a mitogenic role. We also showed by pull-down assays that Nab binds directly to Rotund and Squeeze via its conserved C-terminal domain. We propose two mechanisms by which the activation of wg expression by Rotund in the wing hinge is repressed in the distal wing.

The Drosophila gene zfh2 is required to establish proximal-distal domains in the wing disc

Three main events characterize the development of the proximal-distal axis of the Drosophila wing disc: first, generation of nested circular domains defined by different combinations of gene expression; second, activation of wingless (wg) gene expression in a ring of cells; and third, an increase of cell number in each domain in response to Wg. The mechanisms by which these domains of gene expression are established and maintained are unknown. We have analyzed the role of the gene zinc finger homeodomain 2 (zfh2). We report that in discs lacking zfh2 the limits of the expression domains of the genes tsh, nub, rn, dve and nab coincide, and expression of wg in the wing hinge, is lost. We show that zfh2 expression is delimited distally by Vg, Nub and Dpp signalling, and proximally by Tsh and Dpp. Distal repression of zfh2 permits activation of nab in the wing blade and wg in the wing hinge. We suggest that the proximal-most wing fate, the hinge, is specified first and that later repression of zfh2 permits specification of the distal-most fate, the wing blade. We propose that proximal-distal axis development is achieved by a combination of two strategies: on one hand a process involving proximal to distal specification, with the wing hinge specified first followed later by the distal wing blade; on the other hand, early specification of the proximal-distal domains by different combinations of gene expression. The results we present here indicate that Zfh2 plays a critical role in both processes.

A genetic cascade involving klumpfuss, nab and castor specifies the abdominal leucokinergic neurons in the Drosophila CNS.

Identification of the genetic mechanisms underlying the specification of large numbers of different neuronal cell fates from limited numbers of progenitor cells is at the forefront of developmental neurobiology. In Drosophila, the identities of the different neuronal progenitor cells, the neuroblasts, are specified by a combination of spatial cues. These cues are integrated with temporal competence transitions within each neuroblast to give rise to a specific repertoire of cell types within each lineage. However, the nature of this integration is poorly understood. To begin addressing this issue, we analyze the specification of a small set of peptidergic cells: the abdominal leucokinergic neurons. We identify the progenitors of these neurons, the temporal window in which they are specified and the influence of the Notch signaling pathway on their specification. We also show that the products of the genes klumpfuss, nab and castor play important roles in their specification via a genetic cascade.

Bithorax-complex genes sculpt the pattern of leucokinergic neurons in the Drosophila central nervous system

Although the Hox genes are the main factors involved in the generation of diversity along the anterior/posterior body axis of segmented organisms, it is still largely unknown how these genes act in single cells to determine specific traits at precise developmental stages. The aim of this study was to understand the mechanisms by which Hox genes of the Bithorax complex (Bx-C) of Drosophila act to define segmental differences in the ventral nerve cord of the central nervous system. To achieve this, we have focused on the specification of the leucokinin-expressing neurons. We find that these neurons are specified from the same progenitor neuroblast at two different developmental stages: embryonic and larval neurogenesis. We show that genes of the Bx-C acted in postmitotic cells to specify the segment-specific appearance of leucokinergic cells in the larval and adult ventral nerve cord.

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.

Specification of neuronal subtypes by different levels of Hunchback

During the development of the central nervous system, neural progenitors generate an enormous number of distinct types of neuron and glial cells by asymmetric division. Intrinsic genetic programs define the combinations of transcription factors that determine the fate of each cell, but the precise mechanisms by which all these factors are integrated at the level of individual cells are poorly understood. Here, we analyzed the specification of the neurons in the ventral nerve cord of Drosophila that express Crustacean cardioactive peptide (CCAP). There are two types of CCAP neurons: interneurons and efferent neurons. We found that both are specified during the Hunchback temporal window of neuroblast 3-5, but are not sibling cells. Further, this temporal window generates two ganglion mother cells that give rise to four neurons, which can be identified by the expression of empty spiracles. We show that the expression of Hunchback in the neuroblast increases over time and provide evidence that the absolute levels of Hunchback expression specify the two different CCAP neuronal fates.

Temporal and spatial windows delimit activation of the outer ring of wingless in the Drosophila wing

Extracellular signalling molecules play many roles in the development of higher organisms. They are used reiteratively in different tissues and stages, but the response of the receiving cells is controlled in a context dependent manner. The pattern of expression of the signalling molecule Wingless/WNT in Drosophila is extraordinarily complex. We have studied the mechanism that controls its expression and function in the outer ring of the Drosophila wing hinge. Our findings indicate that wingless expression is controlled by a dual mechanism: its initial activation requires the product of zinc finger homeodomain 2 and is subsequently repressed by the product of the gene complex elbow/no ocelli. This tight regulation restricts the activation of wingless temporally and spatially. Later in development, wingless expression is maintained by an autoregulatory loop that involves the product of homothorax. We have analyzed the phenotype of a wingless allelic combination that specifically removes the outer ring, and our results show that Wingless is required to promote local proliferation of the wing base cells. Thus, cell proliferation in the proximal-distal axis is controlled by the sequential activation of wingless in the inner ring and the outer ring at different stages of development.

Multiple roles of the gene zinc finger homeodomain-2 in the development of the Drosophila wing

The gene zfh2 and its human homolog Atbf1 encode huge molecules with several homeo- and zinc finger domains. It has been reported that they play important roles in neural differentiation and promotion of apoptosis in several tissues of both humans and flies. In the Drosophila wing imaginal disc, Zfh2 is expressed in a dynamic pattern and previous results suggest that it is involved is proximal-distal patterning. In this report we go further in the analysis of the function of this gene in wing development, performing ectopic expression experiments and studying its effects in genes involved in wing development. Our results suggest that Zfh2 plays an important role controlling the expression of several wing genes and in the specification of those cellular properties that define the differences in cell proliferation between proximal and distal domains of the wing disc.

Roles of Hox genes in the patterning of the central nervous system of Drosophila

One of the key aspects of functional nervous systems is the restriction of particular neural subtypes to specific regions, which permits the establishment of differential segment-specific neuromuscular networks. Although Hox genes play a major role in shaping the anterior-posterior body axis during animal development, our understanding of how they act in individual cells to determine particular traits at precise developmental stages is rudimentary. We have used the abdominal leucokinergic neurons (ABLKs) to address this issue. These neurons are generated during both embryonic and postembryonic neurogenesis by the same progenitor neuroblast, and are designated embryonic and postembryonic ABLKs, respectively. We report that the genes of the Bithorax-Complex, Ultrabithorax (Ubx) and abdominal-A (abd-A) are redundantly required to specify the embryonic ABLKs. Moreover, the segment-specific pattern of the postembryonic ABLKs, which are restricted to the most anterior abdominal segments, is controlled by the absence of Abdominal-B (Abd-B), which we found was able to repress the expression of the neuropeptide leucokinin. We discuss this and other examples of how Hox genes generate diversity within the central nervous system of Drosophila.