Complex Autism Spectrum Disorder: Structural Determinants of Health and Their Impact on the Diagnosis.

CASE: Emmanuel is a 6.5-year-old boy who was referred to your evaluation clinic for concerns about his social skills and communication. He arrived in the United States (US) 1 year ago after an immigration trajectory that began in Haiti when he was aged 3 years; passed through Mexico, where the family was in various shelters for over a year; and concluded 2 years later, with the family eventually settling in an urban center in the northeastern United States. While in Mexico, the family was living in a camp without access to utilities. They faced significant food insecurity and experienced multiple relocations because of fears of physical safety.Emmanuel's native language is Haitian Creole, but he learned some Spanish during the year spent in Mexico. Now in the United States, he has been enrolled for the last year in the public school system, where he participates in an inclusion English as a Second Language kindergarten classroom. The school has expressed concern about several behaviors including bolting from the classroom, shouting out inappropriately, and taking food from other children's lunches.On initial meeting with a DBP clinician, Emmanuel's parents report that they do not have any concerns at home about his behavior, although they do feel that he "talks less than his 3 older siblings." The 6-person household is currently living in one-room, temporary housing; they deny current food insecurity.As part of his evaluation, you perform an Autism Diagnostic Observation Scale-2 Module 3 in English with the support of an in-person Haitian Creole interpreter. Emmanuel does not make eye contact throughout the evaluation but does respond to your questions in a combination of English and Haitian Creole. He can define the concept of a "friend" but cannot name one of his own friends. He is not able to engage in the demonstration task with words but does use gestures to indicate the actions involved in brushing teeth. His free play is perseverative and centers around fighting between the action figures.Brief cognitive testing reveals normal nonverbal intelligence. He is unable to decode in English on achievement testing. The family completes a Social Responsiveness Scale in English, which shows normal scores except in the repetitive behaviors section, where the family endorses pacing and some restricted interests, particularly around video games.He is not yet on an Individualized Education Plan, and there have been no formal assessments from the school except for language dominance testing indicating that his dominant language is Haitian Creole, with emerging English skills. What specific topics are unique to the evaluation for autism in an English language learner with a significant trauma history? What factors should be considered when assessing a child with a history of immigration trauma?

Provider and Caregiver Perspectives on Telehealth Assessments for Autism Spectrum Disorder in Young Children: A Multimethod DBPNet Study Exploring Equity.

BACKGROUND: Telehealth uptake increased dramatically during the COVID-19 pandemic, including for autism spectrum disorder (ASD) assessment by developmental-behavioral pediatric (DBP) clinicians. However, little is known about the acceptability of telehealth or its impact on equity in DBP care. OBJECTIVE: Engage providers and caregivers to glean their perspectives on the use of telehealth for ASD assessment in young children, exploring acceptability, benefits, concerns, and its potential role in ameliorating or exacerbating disparities in access to and quality of DBP care. METHODS: This multimethod study used surveys and semistructured interviews to describe provider and family perspectives around the use of telehealth in DBP evaluation of children younger than 5 years with possible ASD between 3/2020 and 12/2021. Surveys were completed by 13 DBP clinicians and 22 caregivers. Semistructured interviews with 12 DBP clinicians and 14 caregivers were conducted, transcribed, coded, and analyzed thematically. RESULTS: Acceptance of and satisfaction with telehealth for ASD assessments in DBP were high for clinicians and most caregivers. Pros and cons concerning assessment quality and access to care were noted. Providers raised concerns about equity of telehealth access, particularly for families with a preferred language other than English. CONCLUSION: This study's results can inform the adoption of telehealth in DBP in an equitable manner beyond the pandemic. DBP providers and families desire the ability to choose telehealth care for different assessment components. Unique factors related to performing observational assessments of young children with developmental and behavioral concerns make telehealth particularly well-suited for DBP care.

Developmentally-Trained Primary Care Clinicians: A Pipeline to Improved Access?

OBJECTIVE: The purpose of this study is to decrease wait time and improve access to developmental-behavioral pediatric (DBP) evaluation in children 4 years of age and younger as part of a quality improvement (QI) initiative in an urban safety-net hospital. METHODS: A primary care pediatrician received DBP minifellowship training 6 hours per week for 1 year to become a developmentally-trained primary care clinician (DT-PCC). DT-PCCs then conducted developmental evaluations that consisted of using a Childhood Autism Rating Scale and Brief Observation of Symptoms of Autism to evaluate children 4 years and younger referred within the practice. Baseline standard practice involved a 3-visit model: DBP advanced practice clinician (DBP-APC) intake visit, neurodevelopmental evaluation by a developmental-behavioral pediatrician (DBP), and feedback by a developmental-behavioral pediatrician. Two QI cycles were completed to streamline the referral and evaluation process. RESULTS: Seventy patients with a mean age of 29.5 months were seen. The average days to initial developmental assessment decreased from 135.3 days to 67.9 days with a streamlined referral to the DT-PCC. Of the 43 patients who required further evaluation by a DBP, the average days to developmental assessment reduced from 290.1 to 120.4 days. CONCLUSION: Developmentally-trained primary care clinicians allowed for earlier access to developmental evaluations. Further research should explore how DT-PCCs can improve access to care and treatment for children with developmental delays.

Access to Care After Autism Diagnosis During the COVID-19 Pandemic: A Quality Improvement Project.

OBJECTIVE: The aim of this study was to identify barriers and improve access to services for children diagnosed with autism spectrum disorder (ASD) during the COVID-19 pandemic. METHODS: Sixty-two patients diagnosed with ASD between March 23 and June 30, 2020, at a large urban safety net hospital were identified by chart review. Patients were called from January to March 2021 and queried regarding access to services. Brief interventions were provided as part of a modified Plan-Do-Study-Act approach. A second chart review was conducted between April and May 2021 to determine whether families accessed any additional services. RESULTS: At the time of initial evaluation, 12 patients (19%) had been able to access all recommended services, 32 (52%) had been able to access some recommended services, and 9 (15%) were not able to access any services. Service levels for 9 families (15%) were undetermined. Sixteen patients (26%) lacked any follow-up with their developmental behavioral pediatrician since their initial evaluation. Thirty patients (48%) reported changes to their individualized educational program or early intervention services, and 17 patients (27%) were receiving in-home applied behavior analysis. After the brief intervention, 13 patients had accessed new services, and 9 patients previously lost to follow-up had completed an appointment. CONCLUSION: Patients diagnosed with ASD in the spring of 2020 had significant difficulty accessing community services. The transition to telemedicine exacerbated this hospital's difficulty contacting patients for follow-up. Additional research is needed to fully characterize the disruptions to services experienced by patients diagnosed with ASD during the pandemic and to increase accessibility and equity of care.

Three-dimensional microCT imaging of mouse heart development from early post-implantation to late fetal stages.

Comprehensive detailed characterization of new mouse models can be challenging due to the individual focus involved in developing these models. Often models are engineered to test a specific hypothesis in a limited number of tissues, stages, and/or other contexts. Whether or not the model produces the desired phenotypes, phenotyping beyond the desired context can be extremely work intensive and these studies are often not undertaken. However, the general information resulting from broader phenotyping can be invaluable to the wider scientific community. The International Mouse Phenotyping Consortium (IMPC) and its subsidiaries, like the Knockout Mouse Project (KOMP), has made great strides in streamlining this process. In particular, the use of microCT has been an invaluable resource in examining internal organ systems throughout fetal/developmental stages. Here, we provide several novel vignettes demonstrating the utility of microCT in uncovering cardiac phenotypes both based on human disease correlations and those that are unpredicted.

Pilot Survey: Police Understanding of Autism Spectrum Disorder.

This piliot study assessed the experiences of police officers related to persons with Autism Spectrum Disorder (ASD) via a survey assessing prior experience, comfort, knowledge, and ability to identify autistic persons. Fifty-one officers completed the survey: 52.9% reported previous ASD training, 34.8% reported personal experience with ASD, and 56.9% endorsed low overall knowledge of ASD. Officers reported neutral comfort (mean 3.24) and moderate practical knowledge (mean 3.74) [Likert scale 1-5; 5 = highest]. Those with previous training or with personal experience reported higher comfort and knowledge. Those with personal experience were more likely to recognize features of ASD in clinical vignettes. Further study is necessary to understand what additional training about ASD may be helpful to police officers.

Raising the Question: Medication Mix-Up or Diversion.

CASE: Max is an 8-year-old boy with autism spectrum disorder and long-standing challenges with sleep maintenance, the latter of which persist despite behavioral intervention and environmental modification. When Max wakes in the early morning hours, he tends to wander the house, which causes his mother to be awake to monitor his safety. Given the impact of Max's fragmented sleep on his functioning and that of his family, you begin a trial of gabapentin liquid to promote sleep maintenance. Soon after, Max's mother reports that he is sleeping through the night, for the first time in his life.Two months later, you receive a message from Max's mother requesting an early refill of his 90-day supply because of having spilled the bottle. You provide a new prescription, and Max's insurance company allows the early refill. Six weeks after that, Max's mother calls to say that she needs another gabapentin prescription because Max has run out. You confirm that she is giving the prescribed dose but are unsure as to why Max is out of medication weeks early. Given these events, you begin to question whether Max's gabapentin prescription is being diverted. What would you do next?

Development of a Training Curriculum for Hospital Security About Autism Spectrum Disorder.

OBJECTIVE: To evaluate whether a newly developed autism spectrum disorder (ASD)-training curriculum for hospital security officers improves comfort, knowledge, and practice. METHODS: Participants were security officers. The ASD-training curriculum was a 45-minute interactive session, adapted from trainings developed for other types of hospital providers. The curriculum included information regarding the presentation of, and challenges faced by, children with ASD in the hospital setting. Officers completed surveys before (T1), immediately after (T2), and 3 months after (T3) the training to assess comfort, knowledge, and practice. Comfort (Likert scale 1-5; 5 = highest) and knowledge (11 yes/no questions) questions were adapted from previous measures. Application of skills was assessed using case scenarios (at T1, T2, and T3) and with officers' report of using various strategies (at T1 and T3; Likert scale 1-5; 5 = always). Data were analyzed using repeated-measures analysis of variance and a series of paired contrast. RESULTS: For the 114 officers who completed surveys, mean comfort scores significantly increased from T1 to T2 (3.48 vs 3.9; p < 0.05), and these gains were maintained at T3 (4.1). Mean percent correct on knowledge questions significantly increased from T1 to T2 (74.6% vs 84.0%; p < 0.05) and was maintained at T3 (82.9%). Officers reported using 2 ASD-supportive strategies significantly more between T1 and T3: using pictures and written communication and asking the caregivers for advice. CONCLUSION: This newly developed ASD-training curriculum for hospital security officers resulted in an immediate increase of self-reported comfort and demonstrated knowledge with continued gains 3 months after training.

A global Slc7a7 knockout mouse model demonstrates characteristic phenotypes of human lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is an inborn error of cationic amino acid (arginine, lysine, ornithine) transport caused by biallelic pathogenic variants in SLC7A7, which encodes the light subunit of the y+LAT1 transporter. Treatments for the complications of LPI, including growth failure, renal disease, pulmonary alveolar proteinosis, autoimmune disorders and osteoporosis, are limited. Given the early lethality of the only published global Slc7a7 knockout mouse model, a viable animal model to investigate global SLC7A7 deficiency is needed. Hence, we generated two mouse models with global Slc7a7 deficiency (Slc7a7em1Lbu/em1Lbu; Slc7a7Lbu/Lbu and Slc7a7em1(IMPC)Bay/em1(IMPC)Bay; Slc7a7Bay/Bay) using CRISPR/Cas9 technology by introducing a deletion of exons 3 and 4. Perinatal lethality was observed in Slc7a7Lbu/Lbu and Slc7a7Bay/Bay mice on the C57BL/6 and C57BL/6NJ inbred genetic backgrounds, respectively. We noted improved survival of Slc7a7Lbu/Lbu mice on the 129 Sv/Ev × C57BL/6 F2 background, but postnatal growth failure occurred. Consistent with human LPI, these Slc7a7Lbu/Lbu mice exhibited reduced plasma and increased urinary concentrations of the cationic amino acids. Histopathological assessment revealed loss of brush border and lipid vacuolation in the renal cortex of Slc7a7Lbu/Lbu mice, which combined with aminoaciduria suggests proximal tubular dysfunction. Micro-computed tomography of L4 vertebrae and skeletal radiographs showed delayed skeletal development and suggested decreased mineralization in Slc7a7Lbu/Lbu mice, respectively. In addition to delayed skeletal development and delayed development in the kidneys, the lungs and liver were observed based on histopathological assessment. Overall, our Slc7a7Lbu/Lbu mouse model on the F2 mixed background recapitulates multiple human LPI phenotypes and may be useful for future studies of LPI pathology.

Human and mouse essentiality screens as a resource for disease gene discovery.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery.

Collaborating with Families and Law Enforcement Agencies to Improve Outcomes for Individuals with Autism Spectrum Disorder.

Increased visibility of adverse encounters between individuals with autism spectrum disorder (ASD) and law enforcement (LE) has stimulated a dialog among providers. There are a variety of contributing factors to the increase, including the recognized lack of training of LE professionals on the needs of individuals with ASD and the paucity of awareness of resources by the families of these individuals. The aim of this article is to provide insight into developmental-behavioral pediatric professionals, to enhance safety and reduce adverse outcomes for individuals with ASD in schools and the community.

High Resolution Imaging of Mouse Embryos and Neonates with X-Ray Micro-Computed Tomography.

Iodine-contrast micro-computed tomography (microCT) 3D imaging provides a non-destructive and high-throughput platform for studying mouse embryo and neonate development. Here we provide protocols on preparing mouse embryos and neonates between embryonic day 8.5 (E8.5) to postnatal day 4 (P4) for iodine-contrast microCT imaging. With the implementation of the STABILITY method to create a polymer-tissue hybrid structure, we have demonstrated that not only is soft tissue shrinkage minimized but also the minimum required time for soft tissue staining with iodine is decreased, especially for E18.5 to P4 samples. In addition, we also provide a protocol on using commercially available X-CLARITYTM hydrogel solution to create the similar polymer-tissue hybrid structure on delicate early post-implantation stage (E8.5 to E14.5) embryos. With its simple sample staining and mounting processes, this protocol is easy to adopt and implement for most of the commercially available, stand-alone microCT systems in order to study mouse development between early post-implantation to early postnatal stages. © 2019 by John Wiley & Sons, Inc.

Bi-allelic Variants in TONSL Cause SPONASTRIME Dysplasia and a Spectrum of Skeletal Dysplasia Phenotypes.

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.

Live imaging of developing mouse retinal slices.

BACKGROUND: Ex vivo, whole-mount explant culture of the rodent retina has proved to be a valuable approach for studying retinal development. In a limited number of recent studies, this method has been coupled to live fluorescent microscopy with the goal of directly observing dynamic cellular events. However, retinal tissue thickness imposes significant technical limitations. To obtain 3-dimensional images with high quality axial resolution, investigators are restricted to specific areas of the retina and require microscopes, such as 2-photon, with a higher level of depth penetrance. Here, we report a retinal live imaging method that is more amenable to a wider array of imaging systems and does not compromise resolution of retinal cross-sectional area. RESULTS: Mouse retinal slice cultures were prepared and standard, inverted confocal microscopy was used to generate movies with high quality resolution of retinal cross-sections. To illustrate the ability of this method to capture discrete, physiologically relevant events during retinal development, we imaged the dynamics of the Fucci cell cycle reporter in both wild type and Cyclin D1 mutant retinal progenitor cells (RPCs) undergoing interkinetic nuclear migration (INM). Like previously reported for the zebrafish, mouse RPCs in G1 phase migrated stochastically and exhibited overall basal drift during development. In contrast, mouse RPCs in G2 phase displayed directed, apical migration toward the ventricular zone prior to mitosis. We also determined that Cyclin D1 knockout RPCs in G2 exhibited a slower apical velocity as compared to wild type. These data are consistent with previous IdU/BrdU window labeling experiments on Cyclin D1 knockout RPCs indicating an elongated cell cycle. Finally, to illustrate the ability to monitor retinal neuron differentiation, we imaged early postnatal horizontal cells (HCs). Time lapse movies uncovered specific HC neurite dynamics consistent with previously published data showing an instructive role for transient vertical neurites in HC mosaic formation. CONCLUSIONS: We have detailed a straightforward method to image mouse retinal slice culture preparations that, due to its relative ease, extends live retinal imaging capabilities to a more diverse group of scientists. We have also shown that, by using a slice technique, we can achieve excellent lateral resolution, which is advantageous for capturing intracellular dynamics and overall cell movements during retinal development and differentiation.

Genome engineering: Drosophila melanogaster and beyond.

A central challenge in investigating biological phenomena is the development of techniques to modify genomic DNA with nucleotide precision that can be transmitted through the germ line. Recent years have brought a boon in these technologies, now collectively known as genome engineering. Defined genomic manipulations at the nucleotide level enable a variety of reverse engineering paradigms, providing new opportunities to interrogate diverse biological functions. These genetic modifications include controlled removal, insertion, and substitution of genetic fragments, both small and large. Small fragments up to a few kilobases (e.g., single nucleotide mutations, small deletions, or gene tagging at single or multiple gene loci) to large fragments up to megabase resolution can be manipulated at single loci to create deletions, duplications, inversions, or translocations of substantial sections of whole chromosome arms. A specialized substitution of chromosomal portions that presumably are functionally orthologous between different organisms through syntenic replacement, can provide proof of evolutionary conservation between regulatory sequences. Large transgenes containing endogenous or synthetic DNA can be integrated at defined genomic locations, permitting an alternative proof of evolutionary conservation, and sophisticated transgenes can be used to interrogate biological phenomena. Precision engineering can additionally be used to manipulate the genomes of organelles (e.g., mitochondria). Novel genome engineering paradigms are often accelerated in existing, easily genetically tractable model organisms, primarily because these paradigms can be integrated in a rigorous, existing technology foundation. The Drosophila melanogaster fly model is ideal for these types of studies. Due to its small genome size, having just four chromosomes, the vast amount of cutting-edge genetic technologies, and its short life-cycle and inexpensive maintenance requirements, the fly is exceptionally amenable to complex genetic analysis using advanced genome engineering. Thus, highly sophisticated methods developed in the fly model can be used in nearly any sequenced organism. Here, we summarize different ways to perform precise inheritable genome engineering using integrases, recombinases, and DNA nucleases in the D. melanogaster. For further resources related to this article, please visit the WIREs website.

A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3' untranslated region-mediated gene regulation.

Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3' untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3' UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3' UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome--a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3' UTR of interest. The pBUTR (piggyBac-based 3' UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.

Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila.

Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, we show that cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. We also observe precocious non-cell autonomous differentiation in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth.

Second chromosome genes required for heart development in Drosophila melanogaster.

Heart development is an evolutionarily conserved process. The cardiac organ of Drosophila melanogaster is the dorsal vessel, a linear contractile tissue with cellular and morphogenetic similarities to the primitive heart tube formed at an early stage of vertebrate heart formation. Abundant evidence shows comparable intercellular signaling pathways and transcription factor networks are utilized in Drosophila and vertebrates, to specify cardiac progenitor cells and instruct their differentiation and function in forming the mature heart. With this proven conservation in mind, we screened the second chromosome of Drosophila for genetic intervals that harbor additional loci required for normal dorsal vessel morphogenesis. Our studies identified numerous regions, that when deleted, culminated in dorsal vessels with abnormal cell numbers and/or structural properties. Certain of the deficiency intervals were further characterized to identify individual genes essential for proper cardiac organ formation. Our analyses identified eight genes of diverse functions that are needed for dorsal vessel development. Several of these sequences have known vertebrate homologues, further supporting a conserved genetic basis for heart formation in Drosophila and higher eukaryotes.

folded gastrulation, cell shape change and the control of myosin localization.

The global cell movements that shape an embryo are driven by intricate changes to the cytoarchitecture of individual cells. In a developing embryo, these changes are controlled by patterning genes that confer cell identity. However, little is known about how patterning genes influence cytoarchitecture to drive changes in cell shape. In this paper, we analyze the function of the folded gastrulation gene (fog), a known target of the patterning gene twist. Our analysis of fog function therefore illuminates a molecular pathway spanning all the way from patterning gene to physical change in cell shape. We show that secretion of Fog protein is apically polarized, making this the earliest polarized component of a pathway that ultimately drives myosin to the apical side of the cell. We demonstrate that fog is both necessary and sufficient to drive apical myosin localization through a mechanism involving activation of myosin contractility with actin. We determine that this contractility driven form of localization involves RhoGEF2 and the downstream effector Rho kinase. This distinguishes apical myosin localization from basal myosin localization, which we find not to require actinomyosin contractility or FOG/RhoGEF2/Rho-kinase signaling. Furthermore, we demonstrate that once localized apically, myosin continues to contract. The force generated by continued myosin contraction is translated into a flattening and constriction of the cell surface through a tethering of the actinomyosin cytoskeleton to the apical adherens junctions. Our analysis of fog function therefore provides a direct link from patterning to cell shape change.

Allocation and specification of the genital disc precursor cells in Drosophila.

The adult structures of Drosophila melanogaster are derived from larval imaginal discs, which originate as clusters of cells within the embryonic ectoderm. The genital imaginal disc is composed of three primordia (female genital, male genital, and anal primordia) that originate from the embryonic tail segments A8, A9, and A10, respectively, and produce the sexually dimorphic genitalia and analia. We show that the genital disc precursor cells (GDPCs) are first detectable during mid-embryogenesis as a 22-cell cluster in the ventral epidermis. Analysis of mutant and double mutant phenotypes of embryonic patterning genes in the GDPCs, together with their expression patterns in these cells, revealed the following with respect to the origins and specification of the GDPCs. The allocation of the GDPCs from the ventral epidermis requires the function of ventral patterning genes, including the EGF receptor and the spitz group of genes. The ventral localization of the GDPCs is further restricted by the action of dorsal patterning genes. Along the anterior-posterior axis, several segment polarity genes (wingless, engrailed, hedgehog, and patched) are required for the proper allocation of the GDPCs. These segment polarity genes are expressed in some, but not all of the GDPCs, indicating that anterior and posterior compartments are not fully established in the GDPCs. In addition, we found that the three primordia of the larval genital disc have already been specified in the GDPCs by the coordinated actions of the homeotic (Hox) genes, abdominal-A, Abdominal-B, and caudal. By identifying how these different patterning networks regulate the allocation and primordial organization of the 22 embryonic precursors of the compound genital disc, we demonstrate that at least some of the organization of the larval disc originates as positional information in the embryo, thus providing a context for further studies on the development of the genital disc.