BioEssays Vol. 6, No. 2 77

Insect Developmental Genetics - Moving Beyond Drosophila Rob Denell

In the past few years, the powerful combination of genetic, developmental and molecular methodologies that can be applied to the fruit fly, Drosophila melanogaster, has resulted in dramatic gains in our understanding of the organization and roles of genes which control early embryological events. A great deal of excitement has been focused on two general classes: ‘seg- mentation’ genes functioning in the periodic organization of the embryo along the anterior-posterior axis, and ‘ homeotic’ genes responsible for the development of segment-specific fea- tures. These genes are clearly involved in complex regulatory interactions, and still greater interest has been sparked by the recognition that the most important homeotic genes and at least three of the segmentation genes include a short region of sequence homology, called the homeo box, that appears to encode a DNA-binding domain, a finding consis- tent with the view that protein products act as transregulatory factors. Homeo boxes have been well conserved evolu- tionarily, and mammals have those of both the Antennapedia-type (character- istic largely of homeotic genes) and the engrailed-type (characteristic of a seg- mentation gene). Because the region of sequence homology is generally restric- ted to the homeo box itself, there is (as chronicled recently on the pages of BioEssays) considerable uncertainty as to the developmental significance of these vertebrate genes.’.

The Diptera (flies) are highly special- ized insects, and this raises another interesting evolutionary question, namely the extent to which the charac- teristics of these Drosophila genes reflect this organism’s phylogenetic history. I will briefly summarize some of the interesting aspects of this question, and then describe new experimental approaches underway in several labora- tories which will result in a broader view of the genetic control of developmental decisions in insects. The discerning reader will note that this particular account would be better entitled ‘What the papers are going to say.’

One of the dominant features of insect morphology is their metameric organ-

ization. It is believed that primitive insects were comprised of 6 head seg- ments, 3 thoracic, and 11 or more abdominal segments. The evolution of the Diptera has included two major modifications: the loss and/or fusion of head and of posterior segments, and the divergence of segments by acquisition of distinctive morphological features. Thus adult flies are highly specialized with respect to their head and dorsal thorax, in which the second segment is relatively very large and bears the flight wings whereas the first and third thoracic segments are much reduced.

Drosophila are also very advanced with respect to some aspects of embryo- genesis. In insects, the ‘germ band’ lies along the ventral surface of the early embryo and gives rise to the mesoderm, nervous system, and external cuticle. As reviewed by Klaus S a n d e ~ , ~ the process by which the germ band becomes segmented varies considerably. At one extreme are insects showing ‘short- germ’ development, in which the germ band initially consists of a head primor- dium and a small posterior bud which produces progressively more posterior segments over time. This is presumably the more primitive pattern, and is similar to events in annelids and crustaceans. In contrast, Drosophila is an example of a ‘long-germ’ insect in which the entire germ band becomes segmented more-or-less simultaneously. Many other insects show behavior intermediate with respect to these ex- tremes. Sander has suggested that segmentation in short-germ insects is likely to be more ‘regulative’ than for long-germ species. One additional spec- ialization of Dipteran embryogenesis is the occurrence of ‘head involution’. In this impressive morphogenetic reorgan- ization, the head segments move through the presumptive mouth to internal positions, and, except for vestiges surrounding the mouth, the resulting larva is essentially headless.

As reviewed by Peter Gergen else- where in this issue, a number of mutations have been identified in Dros- ophila whch are associated with devel- opmental abnormalities in the process of segmentation. Several of these genes

have been molecularly cloned, and studies of their expression as assessed by in situ hybridization to cytoplasmic mRNAs or by antibodies against their protein products have shown very specific spatial patterns which change with time during embryonic develop- ment. It seems likely that the correct establishment of segmental organiza- tion requires complex interactions be- tween the products of these loci. It will be fascinating to compare the genetic mechanisms in Drosophila with those of more primitive insects, with the simplest hypotheses being that these processes have been considerably modified in this long-germ insect or alternatively that they are fundamentally similar but occur far more quickly. Although the latter hypothesis is consistent with the observation that some segmentation genes show earlier expression in the anterior region of the embryo than the posterior (as assayed by in situ hybrid- igation of their RNAs or by immuno- logical screens for the protein products), this is still very much an open question.

With respect to the Drosophila ho- meotic genes, there is now general consensus that two well-separated gene clusters, the bithorax complex (BX-C) and the Antennapedia complex (ANT-C), play primary roles in the establishment of regional identity in embryonic cells and its clonal trans- mission and eventual expression. Edward Lewis, responsible for pioneer- ing work in this field, has long main- tained that this unusual clustering of related genes itself reflects the evolu- tionary history of the Diptera. Loss- of-function mutations of the BX-C cause anteriorly directed homeotic changes of the posterior thorax and abdomen, and the physical order of these genes is correlated with the anterior-posterior sequence of the seg- ments predominantly affected.* Lewis has suggested that the genes responsible for such segment-specific features arose by duplication and divergence of func- tion, thus accounting for their proxim- ity. Although some ANT-C mutations cause homeotic changes of more anter- ior segments, other genes in the complex

78 BioEssays Vol. 6, No. 2

WHAT THE PAPERS SAY mas pas , eu

Fig, I. Linkage relutionshp ,for the Jiue original HOM-C mutations of Tribolium castaneum are depicted, and the segment affected by each is indicated with reference to a druwinx of a mature larva.

are not obviously homeotic and a correlation between chromosomal order and developmental domain is less o b v i o ~ s ; ~ these complexities may be due in part to the advanced nature of anterior development in the Diptera. The opportunity to compare the organ- ization and function of these Drosophila complexes with mechanisms controlling developmental decisions in other insects should provide important insights into those features which are characteristic of the class as a whole and those which are specialized adaptations of the Diptera.

Another interesting area for phylo- genetic comparison is the domains within which the homeotic genes are regulated. Studies of clonal relation- ships during Drosophila development have indicated that embryonic cells become committed not only to particu- lar segmental identities, but to anterior or posterior ‘compartments’ within each segment as well. Studies of mutant phenotypes and the distribution of transcripts and encoded proteins have suggested that BX-C and possibly some ANT-C genes are regulated within regions (called parasegments) comprised of the posterior compartment of one segment and the contiguous anterior compartment of a neighboring seg- rner~t.~ Moreover, the visible embry- onic folds traditionally associated with segmental borders have been interpret- ed as parasegmental borders instead.8 These concepts are still being evaluated in Drosophila, and the assessment of clonal relationships and spatial domains of gene expression in other insects will provide still more interesting aspects of this question.

We are now entering a period in which studies with other insects can meaningfuIIy address some of the ques- tions I have raised. One approach, similar to that being directed towards mammals, is to isolate and study homeo box-containing genes of other insects.

In Walter Gehring’s laboratory in Basel, Uwe Walldorf and Richard Fleig have cloned a number of such genes from a genomic library of the honey bee (a long-germ insect which probably evolved this trait independently from the Dipterans). Sequences flanking the homeo boxes have allowed them to recognize homologies to the Deformed, Sex combs reduced, and possibly An- tennapedia genes of the ANT-C, infra- abdominal-2 of the BX-C, and the segmentation gene engrailed. These observations provide clear evidence of evolutionary relatedness and presu- mably functional similarity. A manu- script comparing the engrailed sequences of Drosophila, bee, mouse and man is presently in preparation. Moreover, preliminary work with a clone homologous to Deformed shows that in situ hybridization to the cyto- plasmic mRNAs of embryos can be used effectively to examine the spatial pattern of expression of these genes.

In Lynn Riddiford’s laboratory in Seattle, Lisa Nagy is taking a similar approach using the tobacco hornworm, Manduca sexta, a moth which has been extensively utilized for physiological and biochemical studies. Nagy has molecularly cloned several restriction fragments showing homology to the Antennapedia homeo box. She is pres- ently studying a 7 kb fragment with respect to its degree of relatedness to Drosophila genes and transcriptional behavior. Similar studies are undoubt- edly underway in other laboratories as well. Future studies of the temporal and spatial specificities of expression of these insect genes are an exciting prospect.

A third insect system now being studied is the red flour beetle, Tribolium castaneum. This organism is interme- diate with respect to germ-band beha- viour and has relatively primitive head organization, with well-defined anten- nae, mandibles, and maxillary and labial appendages in the larva. Richard Beeman of the U.S. Grain Marketing Research Laboratory in Manhattan, Kansas has recently performed com- plementation and recombinational mapping with the eight homeotic mutations available for this organi~m.~ They define six genes, of which five map into a single cluster (denoted the Homeotic complex or HOM-C) on the second linkage group. These recessive mutations affect segments aiong the entire anterior-posterior axis. Maxillo- pedia (mxp) causes the maxilla and labium to develop thoracic legs. In alate prothorax (apt) there is a partial

transformation of the first thoracic segment (Tl) to the second (T2), and (for strong alleles) a reiteration of the elytra (wing covers) characteristic of the latter. Beetles homozygous for missing abdominal sternite (mas) lack ventral structures from the first three ab- dominal segments, whereas pointed abdominal sternite @as) and extra urogomphi (eu) are associated with anterior transformations of the middle and caudal abdominal segments, respec- tively. Most fascinating is the obser- vation that these mutations map on the chromosome in the same order as that of the segments affected (Fig. 1). These results suggest that the HOM-C rep- resents the juxtaposed equivalent of the ANT-C and BX-C. We can speculate that a single homeotic complex rep- resents a more primitive condition, and that during the evolution of the Diptera the ANT-C has become spatially sep- arated and achieved the greater organ- izational complexity and functional diversity which distinguish it from the

Using Drosophila studies as a para- digm, Beeman and I are collaborating on an integrated developmental genetic and molecular characterization of the HOM-C. One important approach will be to saturate the complex with muta- tions associated with visible adult phenotypes or recessive lethality. As a first step, Beeman constructed a viable and fertile stock homozygous for four HOM-C mutations. A cross of irradi- ated wild type beetles to this stock allowed the recognition in the F, of new mutations which fail to complement the existing variants to give visible adult phenotypes. He isoiated a total of 25 new mutations from approximately 39,000 progeny. In addition to recessive mutant alleles, these include eleven dominants affecting the posterior head and T1 (Gsi), T2 and T3 (Ble), or the middle abdominal segments (N). An additional dominant, Skl, shows an interesting discontinuous domain; in heterozygous beetles the first and second abdominal sternites are missing and the eighth abdominal segment is transformed to resemble the seventh. We are attempting to ascertain which of these are genetic inactivations (and possible deficiencies) as opposed to gain-of-function dominants which are common in the Drosophila complexes. Future mutagenesis efforts will be aimed at using a deficiency to saturate the complex with recessive lethal muta- tions. All mutations will also be ex- amined for their effects on earlier stages of the life cycle, with particular empha-

BX-C.

BioEssays Vol. 6, No. 2 79

WHAT THE PAPERS SAY

. . trol of wing disc development in Drosophila. In Cell Patterning (ed. S . Brenner). Asso- ciated Scientific Publishers. Amsterdam, Oxford, New York. Ciba Foundation Sym-

sis on those which result in late embryonic or larval lethality. First instar larvae have thoracic legs and a variety of head features lacking in Drosophila. These structures as well as a wealth of setae and sense organs provide segment-specific markers which will be extremely valuable in scoring homeotic transformations (e.g. see Fig. 2) . In addition, we have shown that the Triboliuni genome includes several restriction fragments with sequence homology to the Antennapedia-type homeo box. We are currently testing to see if Tribolium has homology to the non-homeo box regions of Antenna- pedia and Ultrabithorax cDNAs. In the future we intend to molecularly clone genes recognized by one or both criteria to test the hypothesis that they are located in the HOM-C and to allow a comparison to their putative Drosophila homologues.

In the next few years, the approaches I have described for these and other insects should provide considerable insight into mechanisms controlling segmentation and developmental fate throughout this class.

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Acknowledgements

I wish to thank Walter Gehring, Lisa Nagy, and Richard Beeman for commu- nicating results prior to publication.

REFERENCES 1 WILKINS, A. S. (1986). Homeo box fever, extrapolation and developmental biology. BioEssays 4, 147-148. 2 GEHRING, W. J. (1986). On the homeobox and its significance. BioEssays 5, 3-4.

Fig. 2. This photomicrograph (taken with Nomarski optics) shows aJirst instar larva homozygous for the mutation GsP. In this specimen the labial palps (indicated by large arrowheads) are transformed into antennae; the normal antennae are indicated by small arrowheads. In addition, the posterior head and the prothorax are fused to form a single cephalothorax. Both figures were provirled by Richard Beeman.

3 SANDER, K. (1976). Specification of the basic body pattern in insect embryogenesis. Adv. Insect Physiol. 12, 125-238. 4 LEWIS, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 216, 565, 570. 5 KAUFMAN, T. C. (1983). The genetic regulation of segmentation in Drosophila melanogaster. In Time, Space, and Pattern in Embryonic Development (ed. W. R. Jeffrey & R. A. Raff), pp. 365-383. Alan R. Liss, New York. 6 GARCIA-BELLIDO, A. (1975). Genetic con-

7 MAHOWALD, A . P . & HARDY, P . A . (1985). Genetics of Drosophila embryogen- esis. Annu. Rev. Genetics 19, 149-177. 8 MARTINEZ-ARIAS, A. & LAWRENCE, P. A. (1985). The parasegment and compartments in the Drosophila embryo. Nature 313,

9 BEEMAN, R. W. (1986). A homeotic gene cluster in the red flour beetle. (manuscript submitted to Science.)

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Growth Factors and Their Receptors: Specific Roles in Development Antony W. Burgess

The control of cell movement and proliferation during development has fascinated biologists for most of this century. Many schemes have beer, devised to account for the morpho- genic signals, the precise pathways followed by migrating cells (or growing neurites) and the bursts of tissue-specific proliferation. Until recently there have been few molecular clues to the control

of this tissue-specific development. However, just over a year ago two reports appeared that may provide significant clues; these describe DNA sequences encoding EGF-like molecules in two of the homeotic loci of the Drosophila and C. elegans. The Droso- philu Notch gene encodes a polypeptide chain of 2703 amino acids.l The first half of this polypeptide consists of 36 repeats

which show homology to epidermal growth factor (EGF) (Fig. 1 ) . The Notch product is one of the determin- ants effecting the commitment of ventral ectoderm cells to dermoblasts or neuro- blasts (Fig. 2) . Mutations to the Notch locus cause an unacceptable number of neuronal cells to accumulate in the ventral ectoderm. Similarly, the lin-12 gene in C. elegans codes for a polyeptide