Conserved genetic programs in insect and mammalian brain development

In recent years it has become evident that the developmental regulatory genes involved in patterning the embryonic body plan are conserved throughout the animal kingdom. Striking examples are the orthodenticle (otd/Otx) gene family and the Hox gene family, both of which act in the specification of anteroposterior polarity along the embryonic body axis. Studies carried out in Drosophila and mouse now demonstrate that these genes are also involved in the formation of the insect and mammalian brain; the otd/Otx genes are involved in rostral brain development and the Hox genes are involved in caudal brain development. These studies also show that the genes of the otd/Otx family can functionally replace each other in cross-phylum rescue experiments and indicate that the genetic mechanisms underlying pattern formation in insect and mammalian brain development are evolutionarily conserved. BioEssays 21:677–684, 1999. © 1999 John Wiley & Sons, Inc.     

Conserved usage of gap and homeotic genes in patterning the CNS.

The homeotic and cephalic gap genes play central roles in the specification of the anteroposterior animal body axis. Genetic studies carried out in Drosophila and mouse now demonstrate that these genes are also involved in embryonic brain development. The homeotic genes act in posterior brain patterning, and the cephalic gap genes act in anterior brain patterning. Moreover, striking cross-phylum gene replacement experiments show that invertebrate and vertebrate members of the orthodenticle gene family can functionally replace each other. These findings indicate that the genetic mechanisms involved in embryonic brain development are conserved and suggest a common evolutionary origin of the insect and vertebrate brain.     

Insights into the urbilaterian brain: conserved genetic patterning mechanisms in insect and vertebrate brain development

Recent molecular genetic analyses of Drosophila melanogaster and mouse central nervous system (CNS) development revealed strikingly similar genetic patterning mechanisms in the formation of the insect and vertebrate brain. Thus, in both insects and vertebrates, the correct regionalization and neuronal identity of the anterior brain anlage is controlled by the cephalic gap genes otd/Otx and ems/Emx, whereas members of the Hox genes are involved in patterning of the posterior brain. A third intermediate domain on the anteroposterior axis of the vertebrate and insect brain is characterized by the expression of the Pax2/5/8 orthologues, suggesting that the tripartite ground plans of the protostome and deuterostome brains share a common evolutionary origin. Furthermore, cross-phylum rescue experiments demonstrate that insect and mammalian members of the otd/Otx and ems/Emx gene families can functionally replace each other in embryonic brain patterning. Homologous genes involved in dorsoventral regionalization of the CNS in vertebrates and insects show remarkably similar patterning and orientation with respect to the neurogenic region (ventral in insects and dorsal in vertebrates). This supports the notion that a dorsoventral body axis inversion occurred after the separation of protostome and deuterostome lineages in evolution. Taken together, these findings demonstrate conserved genetic patterning mechanisms in insect and vertebrate brain development and suggest a monophyletic origin of the brain in protostome and deuterostome bilaterians.     

Nanos plays a conserved role in axial patterning outside of the Diptera

Axial patterning is a fundamental event in early development, and molecules involved in determining the body axes provide a coordinate system for subsequent patterning. While orthologs of Drosophila bicoid and nanos play a conserved role in anteroposterior (AP) patterning within at least a subset of Diptera, conservation of this process has not yet been demonstrated outside of the flies. Indeed, it has been argued that bicoid, an instrumental "anterior" factor in Drosophila melanogaster, acquired this role during the evolution of more-derived dipterans. Interestingly, the interaction of Drosophila maternal nanos and maternal hunchback provides a system for patterning the AP axis that is partially redundant to the anterior system. Previous studies in grasshoppers suggest that hunchback may play a conserved role in axial patterning in this insect, but this function may be supplied solely by the zygotic component of hunchback expression. Here we provide evidence that the early pattern of zygotic grasshopper Hunchback expression is achieved through translational repression that may be mediated through the action of grasshopper nanos. This is consistent with the notion that an anterior gradient system is not necessary in all insects and that the posterior pole "probably conveys longitudinal polarity on the ensuing germ anlage".     

Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation

While the expression patterns of segment polarity genes such as engrailed have been shown to be similar in Drosophila melanogaster and Schistocerca americana (grasshopper), the expression patterns of pair-rule genes such as even-skipped are not conserved between these species. This might suggest that the factors upstream of pair-rule gene expression are not conserved across insect species. We find that, despite this, many aspects of the expression of the Drosophila gap gene hunchback are shared with its orthologs in the grasshoppers S. americana and L. migratoria. We have analyzed both mRNA and protein expression during development, and find that the grasshopper hunchback orthologs appear to have a conserved role in early axial patterning of the germ anlagen and in the specification of gnathal and thoracic primordia. In addition, distinct stepped expression levels of hunchback in the gnathal/thoracic domains suggest that grasshopper hunchback may act in a concentration-dependent fashion (as in Drosophila), although morphogenetic activity is not set up by diffusion to form a smooth gradient. Axial patterning functions appear to be performed entirely by zygotic hunchback, a fundamental difference from Drosophila in which maternal and zygotic hunchback play redundant roles. In grasshoppers, maternal hunchback activity is provided uniformly to the embryo as protein and, we suggest, serves a distinct role in distinguishing embryonic from extra-embryonic cells along the anteroposterior axis from the outset of development - a distinction made in Drosophila along the dorsoventral axis later in development. Later hunchback expression in the abdominal segments is conserved, as are patterns in the nervous system, and in both Drosophila and grasshopper, hunchback is expressed in a subset of extra-embryonic cells. Thus, while the expected domains of hunchback expression are conserved in Schistocerca, we have found surprising and fundamental differences in axial patterning, and have identified a previously unreported domain of expression in Drosophila that suggests conservation of a function in extra-embryonic patterning.     

Giant, Krüppel, and caudal act as gap genes with extensive roles in patterning the honeybee embryo

In Drosophila, gap genes translate positional information from gradients of maternal coordinate activity and act to position the periodic patterns of pair-rule gene stripes across broad domains of the embryo. In holometabolous insects, maternal coordinate genes are fast-evolving, the domains that gap genes specify often differ from their orthologues in Drosophila while the expression of pair-rule genes is more conserved. This implies that gap genes may buffer the fast-evolving maternal coordinate genes to give a more conserved pair-rule output. To test this idea, we have examined the function and expression of three honeybee orthologues of gap genes, Krüppel, caudal, and giant. In honeybees, where many Drosophila maternal coordinate genes are missing, these three gap genes have more extensive domains of expression and activity than in other insects. Unusually, honeybee caudal mRNA is initially localized to the anterior of the oocyte and embryo, yet it has no discernible function in that domain. We have also examined the influence of these three genes on the expression of honeybee even-skipped and a honeybee orthologue of engrailed and show that the way that these genes influence segmental patterning differs from Drosophila. We conclude that while the fundamental function of these gap genes is conserved in the honeybee, shifts in their expression and function have occurred, perhaps due to the apparently different maternal patterning systems in this insect.     

A dual role for nanos and pumilio in anterior and posterior blastodermal patterning of the short-germ beetle Tribolium castaneum

Abdominal patterning in Drosophila requires the function of Nanos (nos) and Pumilio (pum) to repress posterior translation of hunchback mRNA. Here we provide the first functional analysis of nanos and pumilio genes during blastodermal patterning of a short-germ insect. We found that nos and pum in the red flour beetle Tribolium castaneum crucially contribute to posterior segmentation by preventing hunchback translation. While this function seems to be conserved among insects, we provide evidence that Nos and Pum may also act on giant expression, another gap gene. After depletion of nos and pum by parental RNAi, Hunchback and giant remain ectopically at the posterior blastoderm and the posterior Krüppel (Kr) domain is not being activated. giant may be a direct target of Nanos and Pumilio in Tribolium and presumably prevents early Kr expression. In the absence of Kr, the majority of secondary gap gene domains fail to be activated, and abdominal segmentation is terminated prematurely. Surprisingly, we found Nos and Pum also to be involved in early head patterning, as the loss of Nos and Pum results in deletions and transformations of gnathal and pre-gnathal anlagen. Since the targets of Nos and Pum in head development remain to be identified, we propose that anterior patterning in Tribolium may involve additional maternal factors.     

Cross-phylum regulatory potential of the ascidian Otx gene in brain development in Drosophila melanogaster

The origin of molecular mechanisms of cephalic development is an intriguing question in evolutionary and developmental biology. Ascidians, positioned near the origin of the phylum Chordata, share a conserved set of anteroposterior patterning genes with vertebrates. Here we report the cross-phylum regulatory potential of the ascidian Otx gene in the development of the Drosophila brain and the head vertex structures. The ascidian Otx gene rescued the embryonic brain defect caused by a null mutation of the Drosophila orthodenticle (otd) gene and enhanced rostral brain development while it suppressed trunk nerve cord formation. Furthermore, the ascidian Otx gene restored the head vertex defects caused by a viable otd mutation, ocelliless, via specific activation and repression of downstream regulatory genes. These cross-phylum regulatory potentials of the ascidian Otx gene are equivalent to the activities of the Drosophila and human otd/Otx genes in these developmental processes. These results support the notion that basal chordates such as ascidians have the same molecular patterning mechanism for the anterior structures found in higher chordates, and suggest a common genetic program of cephalic development in invertebrate, protochordate and vertebrate.     

Wasps, beetles and the beginning of the ends

Recent papers investigating the genes regulating early embryogenesis in the wasp Nasonia vitripennis and the beetle Tribolium castaneum have provided us with important clues as to how early development is controlled in insects other than higher dipterans such as Drosophila melanogaster. The results of these studies demonstrate that in insects that do not have bicoid, anterior patterning is regulated by a combination of maternal orthodenticle and hunchback. Furthermore, during the evolution of long-germ-band development, Nasonia and Drosophila may have evolved different mechanisms to pattern posterior segments, marginalising the important role of the terminal system in short-germ-band embryos.     

Analysis of maternal effect mutant combinations elucidates regulation and function of the overlap of hunchback and Krüppel gene expression in the Drosophila blastoderm embryo

The metameric organisation of the Drosophila embryo is generated early during development, due to the action of maternal effect and zygotic segmentation and homeotic genes. The gap genes participate in the complex process of pattern formation by providing a link between the maternal and the zygotic gene activities. Under the influence of maternal gene products they become expressed in distinct domains along the anteroposterior axis of the embryo; negative interactions between neighboring gap genes are thought to be involved in establishing the expression domains. The gap gene activities in turn are required for the correct patterning of the pair-rule genes; little is known, however, about the underlying mechanisms. We have monitored the distribution of gap and pair-rule genes in wild-type embryos and in embryos in which the anteroposterior body pattern is greatly simplified due to combinations of maternal effect mutations (staufen exuperantia, vasa exuperantia, vasa exuperantia, bicoid oskar, bicoid oskar torsolike, vasa torso exuperantia). We show that the domains of protein distribution of the gap genes hunchback and Krüppel overlap in wild-type embryos. Based on the analysis of the maternal mutant combinations, we suggest an explanation of how this overlap is generated. Furthermore, our data show that different constellations of gap gene activities provide different input for the pair-rule genes, and thus strongly suggest that the overlap of hunchback and Krüppel in wild-type is functional in the formation of the patterns of pair-rule genes.     

A caudal mRNA gradient controls posterior development in the wasp Nasonia

One of the earliest steps of embryonic development is the establishment of polarity along the anteroposterior axis. Extensive studies of Drosophila embryonic development have elucidated mechanisms for establishing polarity, while studies with other model systems have found that many of these molecular components are conserved through evolution. One exception is Bicoid, the master organizer of anterior development in Drosophila and higher dipterans, which is not conserved. Thus, the study of anteroposterior patterning in insects that lack Bicoid can provide insight into the evolution of the diversity of body plan patterning networks. To this end, we have established the long germ parasitic wasp Nasonia vitripennis as a model for comparative studies with Drosophila. Here we report that, in Nasonia, a gradient of localized caudal mRNA directs posterior patterning, whereas, in Drosophila, the gradient of maternal Caudal protein is established through translational repression by Bicoid of homogeneous caudal mRNA. Loss of caudal function in Nasonia results in severe segmentation defects. We show that Nasonia caudal is an activator of gap gene expression that acts far towards the anterior of the embryo, placing it atop a cascade of early patterning. By contrast, activation of gap genes in flies relies on redundant functions of Bicoid and Caudal, leading to a lack of dramatic action on gap gene expression: caudal instead plays a limited role as an activator of pair-rule gene expression. These studies, together with studies in short germ insects, suggest that caudal is an ancestral master organizer of patterning, and that its role has been reduced in higher dipterans such as Drosophila.     

Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus

In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, a long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, we have investigated the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb). Here, we have focused on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, we found the following: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of our results with the results for other derived species revealed that the canonical hb function may have evolved from the non-canonical hb functions during evolution.     

The evolution of oocyte patterning in insects: multiple cell-signaling pathways are active during honeybee oogenesis and are likely to play a role in axis patterning

SUMMARY In Drosophila it is well established that signaling between the germline and surrounding follicle cells establishes the axes of the future embryo and is required for patterning of the eggshell. However, little is known about how this is achieved in other insects. Genome sequencing studies imply that maternal axis determination may be rapidly evolving, as a number of Drosophila maternal patterning genes are absent from the genomes of other insects. We have examined the distribution and function of six developmental signaling pathways present, and active, in honeybee ovarioles. We have confirmed an evolutionarily conserved role for transforming growth factor-α-epidermal growth factor receptor signaling in dorsal-ventral (DV) patterning. We also found evidence for the involvement of Dpp/Mad and JNK-MAPK pathways in DV patterning, unlike Drosophila. Several of these pathways are also active in the germarium, implicating them in germ and somatic stem cell maintenance and proliferation, similar to their activities in Drosophila ovaries.     

Distinct mechanisms for mRNA localization during embryonic axis specification in the wasp Nasonia

mRNA localization is a powerful mechanism for targeting factors to different regions of the cell and is used in Drosophila to pattern the early embryo. During oogenesis of the wasp Nasonia, mRNA localization is used extensively to replace the function of the Drosophila bicoid gene for the initiation of patterning along the antero-posterior axis. Nasonia localizes both caudal and nanos to the posterior pole, whereas giant mRNA is localized to the anterior pole of the oocyte; orthodenticle1 (otd1) is localized to both the anterior and posterior poles. The abundance of differentially localized mRNAs during Nasonia oogenesis provided a unique opportunity to study the different mechanisms involved in mRNA localization. Through pharmacological disruption of the microtubule network, we found that both anterior otd1 and giant, as well as posterior caudal mRNA localization was microtubule-dependent. Conversely, posterior otd1 and nanos mRNA localized correctly to the posterior upon microtubule disruption. However, actin is important in anchoring these two posteriorly localized mRNAs to the oosome, the structure containing the pole plasm. Moreover, we find that knocking down the functions of the genes tudor and Bicaudal-D mimics disruption of microtubules, suggesting that tudor's function in Nasonia is different from flies, where it is involved in formation of the pole plasm.     

Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly.