During most of my career I have been interested in the developmental challenges of metamorphosis and the way that hormones orchestrate the underlying cellular and molecular events. In insects, molting and metamorphosis are regulated by two hormones: ecdysone, which causes molting and promotes metamorphosis, and juvenile hormone (JH), which allows larval molting but prevents metamorphosis. The insect epidermis makes the overlying cuticle or exoskeleton and in most insects is polymorphic, in that it must switch genetic programs at the time of metamorphosis to the pupa, then to the adult. Generation of the adult also depends on imaginal discs and primordia that grow during larval life, then differentiate when cued by the hormonal milieu at metamorphosis. Finally, larval-specific structures die at metamorphosis. Internally the central nervous system undergoes metamorphosis by a similar combination of remodeling of some neurons, development of new adult-specific neurons that were generated by neuroblasts during embryonic and larval development, and cell death of larva-specific neurons.

Juvenile hormone, a sesquiterpenoid, plays two roles in maintaining the larval state: (1) It allows the modulation of ongoing gene expression by ecdysone but prevents the activation of new genes and repression of active genes by this steroid hormone that are necessary for switching of genetic programs. (2) It permits proliferative growth but prevents morphogenesis of imaginal discs and primordia. This morphostatic action does not require ecdysteroid. My major goal is to understand the molecular mechanism of action of this unique hormone in these two different roles.

Juvenile Hormone and Metamorphosis
Our studies on the epidermis of Lepidoptera have shown that JH can prevent both the ecdysone-induced appearance of the transcription factor Broad that is necessary for specifying the pupal program and the ecdysone-induced disappearance of Broad that is necessary for the subsequent adult developmental program. In Drosophila, by contrast, JH does not prevent metamorphosis of the imaginal disc–derived structures but can prevent the adult differentiation of the imaginal abdominal epidermis derived from the histoblasts. In this case, JH treatment prevents the normal disappearance of Broad from the abdominal epidermis during adult differentiation, resulting in the formation of a pupal, rather than adult, cuticle.

We find a second key transcription factor is Kruppel homolog 1, which is found primarily in the larva and regulates aspects of the timing of the ecdysone-induced transcription factor cascade at the onset of metamorphosis. It is aberrantly up-regulated by JH in Drosophila and, in turn, regulates the misexpression of broad in parts of the abdominal epidermis. Thus, the regulation of both "switch genes" and temporal and spatial controllers induced by ecdysone can be influenced by JH. We will continue to explore the implications of these findings to the action of JH on the proliferation of the histoblasts to form the imaginal epidermal cells and their replacement of the larval epidermis.

At Janelia Farm, I plan to explore some of the tantalizing actions that we have seen in the effects of JH on the metamorphosis of the central nervous system. First, we found 10 years ago that JH "freezes" the development of the optic lobes of the adult brain at a very early stage in their organization of the medulla columns that are organized by ingrowing photoreceptors. This cessation of development is accompanied by a dramatic shift in the ecdysone receptor isoforms that are normally present at this time. The tools are now available to explore the cellular and molecular aspects of this cessation. Second, preliminary observations show that the ventral nervous system reorganization at metamorphosis is disrupted by JH. We plan to collaborate with James Truman (HHMI, Janelia Farm Research Campus) to generate clones in specific lineages to determine how they may be affected by JH.

Insulin and Growth
Size control is a developmental process that occurs in virtually all living organisms, yet little is known about the mechanisms used to assess size and how they are then processed to control body size. Metamorphosis in the insect, like puberty in humans, occurs when the species-specific size has been attained. Growth of the larva is regulated by the incoming nutrients and mediated by insulin signaling based on this nutritional input. Once a critical weight is reached, endocrine mechanisms come into play to initiate metamorphosis. We and others have recently shown that insulin-dependent growth of the prothoracic glands that produce the ecdysone for molting is involved in the assessment of this critical weight for Drosophila. In this case, surpassing critical weight leads to nutrient-independent differentiation leading to metamorphosis, at least partially controlled by ecdysone-mediated derepression of certain genes and by the disappearance of JH. I plan to continue the study of the nutrient-dependent regulation of the neuroendocrine pathway that culminates in metamorphosis.

A second focus of my laboratory will be a study of the role of nutrition and insulin signaling in controlling neurogenesis and the differentiation of new neurons during larval growth in Drosophila. The neuroblasts form during embryonic development but most then arrest after making an initial set of larval neurons. They then resume proliferation during the first instar and are dependent on incoming nutrients, but the timing of this resumption differs. Is this resumption dependent on insulin signaling? If so, how is it temporally regulated among the neuroblasts? Is it dependent on the presence of JH? Besides looking at neurogenesis, itself, I am currently collaborating with Truman to determine if the changing endocrine environment of the growing larva provides temporal cues that specify different subclasses of secondary neurons during larval life.

Hormonal Basis of the Evolution of Metamorphosis
Several years ago, Truman and I developed a hypothesis that a change in the timing of JH appearance during embryonic development of the direct developing insects may have driven the evolution of complete metamorphosis. We have been using diverse direct developing species such as firebrats, locusts, crickets, the milkweed bug Oncopeltus fasciatus, and the relatively primitive metamorphic flour beetle Triboliumcastaneum, as well as two crustaceans (barnacle and amphipod), to study the effects of JH on embryonic development and on the development of imaginal primordia. We have found that the pupal-specific transcription factor Broad of the more derived metamorphic insects first appears in the embryo in these species. Moreover, initial loss-of-function experiments in Oncopeltus and Tribolium show that Broad is necessary for the progressive morphogenesis and anisometric growth during nymphal life in the direct developing Oncopeltus, but seems to have little function in the beetle until the onset of metamorphosis. I intend to delve further into the evolution of the broad gene and its signaling role as a key to the evolution of metamorphosis.

Hormones and Behavior
My previous studies on hormones and behavior centered on the environmental and neuroendocrine control of adult reproductive behavior of female moths, i.e. the behaviors involved in release of the sex pheromone and in the change from virgin to mated behavior. At Janelia Farm I plan to reactivate these interests, switching to the use of Drosophila. The switch in larvae from feeding to wandering behavior at the onset of metamorphosis depends on exposure to ecdysone in the absence of JH. The switch from virgin to mated behavior in the adult is mediated by various peptides found in the accessory gland secretions of the male. One of these, the sex peptide, also increases JH production by the corpora allata. We will use dominant-negative receptors and RNA interference to suppress the responses to ecdysone and to JH in a cell-autonomous fashion. Using the array of GAL4 lines developed by Gerald Rubin (HHMI, JFRC), I intend to screen for neurons that are involved in these hormonally controlled, behavioral switches.