ICVM-10 Symposium on Reptile Skeletal Biology

ICVMInterested in the latest research on reptile development, biomechanics and evolution? Come to our symposium at the International Congress of Vertebrate Morphology in Barcelona next Tuesday (July 9, 2013). If not, be sure to catch some of the other outstanding talks held in the neighboring rooms. 

Symposium 7: Reptile Skeletal Biology: Investigations Into Tissue Morphology, Development, and Evolution

Organizers: Casey Holliday, University of Missouri; Matthew Vickaryous, University of Guelph

Reptiles are one of the most ancient and morphologically diverse radiations of tetrapods.  An important feature underpinning this diversity is the skeleton. While the reptilian skeleton has a long history of appreciation by palaeontologists, morphologists and ecologists, it is now emerging as an important model for many developmental and biomedical biologists.  Furthermore, the adoption of various cutting edge approaches in molecular, imaging, and experimental techniques is leading to major revisions and re-interpretations of several longstanding ideas.  This symposium will focus on exploring some of the most intriguing and fundamental questions in evolutionary developmental biology from a uniquely reptilian perspective.  Our participants will bring forward important advancements in the study of the origin and evolution of body plans, morphogenesis and regeneration, and physiology and functional morphology . The goal of our assembled international panel (including participants from Japan, Germany, UK, France, Canada and the US) is to provide a productive and collaborative forum to share, critique and exchange approaches, techniques and species-specific expertise.  Building on the recent publication of the Anolis genome and recent funding to complete the Alligator genome, reptilian biology is undergoing an unparalleled renaissance and our symposium will highlight the latest research using turtles, lepidosaurs, crocodylians, and their fossil ancestors.  ICVM-10 presents an exceptional opportunity to highlight this next generation of reptilian skeletal biology, and its ever growing potential for the broader study of development, evolution, and functional morphology.

09:30

S-036 SKELETAL REGENERATION FOLLOWING TAIL LOSS IN LIZARDS

Vickaryous, Matt; Coates, Helen; Delorme, Steph University of Guelph, Guelph, Canada

10:00

S-037 SQUAMATE VERTEBRAL HISTOLOGY AND MICROANATOMY -DEVELOPMENT AND EVOLUTION

Houssaye, Alexandra Steinmann Institut für Geologie, Paläontologie und Mineralogie, Universität Bonn, Bonn, Germany

10:30

S-038 COMPARATIVE SKULL MECHANICS OF THE LIZARDS TUPINAMBIS MERIANAE AND VARANUS ORNATUS

Gröning, Flora (1); Jones, Marc (2); Curtis, Neil (1); O’higgins, Paul (3); Evans, Susan (2); Fagan, Michael (1) (1) University of Hull, Hull, United Kingdom; (2) University College London, London, United Kingdom; (3) University of York, York, United Kingdom

11:00

S-039 A COMPARISON OF TURTLE AND CHICKEN ONTOGENY REVEALS THE BASIS FOR DIVERGENT HARD PALATE MORPHOLOGY

Richman, Joy; Abramyan, John; Leung, KelvinLife Sciences Centre, University of British Columbia, Canada

12:00

S-040 HOW DID ENAMEL MATRIX PROTEINS EVOLVE IN REPTILE TEETH AND ARE THEY PRESENT IN OSTEODERMS?

Sire, Jean-Yves (1); Gasse, Barbara (1); Silvent, Jérémie (1); Delgado, Sidney (1); Belheouane, Meriem (1); De Buffrénil, Vivian (2) (1) Université Pierre et Marie Curie, Paris, France; (2) Muséum national d’Histoire naturelle, Paris, France

12:30

S-041 DEVELOPMENTAL PLAN OF THE AMNIOTE SHOULDER GIRDLE AND ITS EVOLUTIONARY DIVERSITY

Nagashima, Hiroshi (1); Hirasawa, Tatsuya (2); Sugahara, Fumiaki (2); Takechi, Masaki (3); Sato, Noboru (1); Kuratani, Shigeru (2) (1) Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; (2) RIKEN Center for Developmental Biology, Kobe, Japan; (3) Iwate Medical University, Yahaba-cho, Japan

14:30

S-042 MORPHOLOGY AND FUNCTION OF THE REPTILE MANDIBULAR SYMPHYSIS

Holliday, Casey (1); Hieronymus, Tobin (2); Nesbitt, Sterling (3); Vickaryous, Matthew (4) (1) University of Missouri, Columbia, United States; (2) Northeastern Ohio Medical University, Kent, United States; (3) Field Museum of Natural History, Chicago, United States; (4) University of Guelph, Guelph, Canada

15:00

S-043 DEVELOPMENT AND EVOLUTION OF MESOPODIALIZATION IN THE ICHTHYOSAURIAN LIMB SKELETON

Maxwell, Erin (1); Scheyer, TorstenM. (1); Fowler, Donald (2) (1) Universität Zürich, Paläontologisches Institut und Museum, Switzerland; (2) McGill University, Department of Biology, Canada

15:30

S-044 FRONTIERS IN THE EVOLUTION AND DEVELOPMENT OF THE REPTILIAN SKULL

Bhullar, Bhart-Anjan (1); Marugan-Lobon, Jesus (2); Racimo, Fernando (3); Bever, Gabe (4); Rowe, Timothy (5); Norell, Mark (6); Abzhanov, Arhat (1) (1) Harvard University, Cambridge, United States; (2) University of Madrid, Madrid, Spain; (3) University of California, Berkeley, United States; (4) New York College of Osteopathic Medicine, Old Westbury, United States; (5) The University of Texas at Austin, Austin, United States; (6) American Museum of Natural History, New York, United States

16:00

S-045 CONSERVATION OF PRIMAXIAL REGIONALIZATION IN THE EVOLUTION OF THE SNAKE BODY FORM INDICATES HOMOPLASY IN HOX GENE FUNCTION

Head, Jason (1); Polly, P.David (2) (1) University of Nebraska-Lincoln, Lincoln, United States; (2) Indiana University, Bloomington, United States

17:00

S-046 IN VIVO CRANIAL BONE STRAINS DURING FEEDING IN THE LIZARDS TUPINAMBIS AND UROMASTYX

Porro, Laura (1); Ross, Callum (2); Herrel, Anthony (3); Evans, Susan (4); Fagan, Michael (5); O’Higgins, Paul (6) (1) University of Bristol, Bristol, United Kingdom; (2) University of Chicago, Chicago, United States; (3) CNRS/Muséum National d’Histoire Naturelle, Paris, France; (4) University College London, London, United Kingdom; (5) University of Hull, Hull, United Kingdom; (6) University of York, York, United Kingdom

 17:30

S-047 ARCHOSAUROMORPH BONE HISTOLOGY REVEALS EARLY EVOLUTION OF ELEVATED GROWTH AND METABOLIC RATES

Werning, Sarah (1); Irmis, Randall (2); Nesbitt, Sterling (3); Smith, Nathan (4); Turner, Alan (5); Padian, Kevin (1) (1) University of California, Berkeley,CA, United States; (2) Natural History Museum of Utah & University of Utah, Salt Lake City, UT, United States; (3) The Field Museum, Chicago, IL, United States; (4) Howard University, Washington, DC, United States; (5) Stony Brook University, Stony Brook, NY, United States

Towards Finding Invisible Whiskers in Fossil Crocs

Our paper on Alligator Trigeminal Nerve Scaling and Significance is out in Anatomical Record:

http://onlinelibrary.wiley.com/doi/10.1002/ar.22666/pdf

You can catch University of Missouri’s press release here:

https://nbsubscribe.missouri.edu/news-releases/2013/0404-new-measurement-of-crocodilian-nerves-could-lead-to-better-understanding-of-ancient-animals%E2%80%99-behavior-mu-researcher-finds/

and a snazzy video describing the research here:

http://vimeo.com/user2631252

 

This was all part of a lab rotation project by Grad Student and Life Sciences Fellow Ian George. Ian tried his hand at a Blog Post too, take it away Ian:

Croc brains and ganglia

Croc brains and ganglia

How is it that alligators are deadly accurate hunters, even on the darkest nights when their prey makes no sounds? The answer to that are dome pressure receptors, highly sensitive small black dots that pepper the sides of their face, first described by Von During (1974), which act much in the same way as whiskers do on a cat (Soares, 2002; Leitch & Catania, 2012). As soon as an unsuspecting animal disturbs the water to get a drink, these invisible whiskers detect the tiny waves they create and the alligator can strike having neither seen nor heard the animal. While we can see and count these dots in living alligators, what about in their ancestors? When did this specialized sense first appear?

Before we can determine if fossil crocodyliforms may have had these dome pressure receptors (DPRs), which are a soft tissue structure, we first need to look for some feature on the skull associated with them. Just like in humans, the face of the alligator gets its sensation from the trigeminal nerve (CNV). This nerve supplies sensation to the face of all vertebrates and evidence suggests that it is larger when there are specialized receptors present like whiskers or DPRs. This large nerve also has a large hole in the skull associated with it, the trigeminal fossa, which we can measure in living alligators and fossil crocodyliforms. Therefore this hole in the skull is an excellent place to measure the nerve that supplies the DPRs because it is present in both living alligators as wells preserved in fossils.

Alligator trigeminal nerve

Alligator trigeminal nerve

Our recent article in the Anatomical Record explores the trigeminal DPR system through an anatomical investigation of a range of different sized alligators, a few crocodiles and some fossil crocodyliforms. We CT scanned alligator heads to get volumetric measurements, dissected them to better understand the anatomy of the trigeminal nerve, and finally histologically sampled the nerve to measure how many fibers were in it. This latter part helped distinguish the relative contributions of motor versus sensory portions of the nerve. Our findings show that the trigeminal nerve scales with skull size in the alligator as well as with brain size, an important factor when measuring nervous tissue. Together with data we took from the skulls of fossilized crocodyliforms comparing the relative size of the trigeminal fossa and the maxillomandibular foramen in the skull to the overall size of the skull and brain, we can now get a good idea of relative face touch. Integrating these data with perhaps integumentary osteological correlates may then give us a good idea about DPR evolution.

This important new tool can give us new information about the habitat these extinct crocodyliforms may have lived in. If the trigeminal fossa in an extinct croc is substantially smaller than that of a living crocodylian, it may not have had DPRs or at least have had a less-sensitive face. This has bearing on the animal’s relationship with its environment and certainly can be applied to other taxa that have well encrusted trigeminal fossae.

Leitch DB, Catania KC. 2012. Structure, innervation and response properties of integumentary sensory organs in crocodilians. Journal of Experimental Biology 215:4217–4230.

Soares D. 2002. An ancient sensory organ in crocodilians. Nature 417:241–242.

von During M. 1974. The ultrastructure of the cutaneous receptors in the skin of Caiman crocodilus. Abhandlungen Rhein.-Westfal. Akad 53:123–134.

 

A Brief History of Archosauriform Symphyses

Class-based evolution of the mandibular symphysis of archosauriforms. Holliday and Nesbitt, 2013.

Class-based evolution of the mandibular symphysis of archosauriforms. Holliday and Nesbitt, 2013.

Two years ago, I was invited to present in the Basal Archosaur symposium at the Latin American Congress of Vertebrate Paleontology in San Juan, Argentina. Holy cow do they know how to put on a good conference.  This symposium has turned into a Special Issue of the Geological Society of London. Papers in this volume will be trickling out over the next few months I presume. Ours is out now though. Sterling and I saw it as a good opportunity to begin a larger project on the functional significance and evolution of chins in archosaurs. Stemming from my earlier work on lizard symphyses (which we still have unpublished data of), ongoing work on crocodyliform chins, and a general interest in cranial arthrology, this paper presents the broad picture of diversity of symphyses among early archosaurs and up into the crown groups. From reading the paper, I hope people will take home just how incredibly diverse the chin, and feeding apparatus really is among this exciting group of vertebrates, and how often members of the larger group have experimented with convergent morphotypes. I think this is evidenced by the colorful character tree to the right (Fig. 12 from the paper).

One thing that I’ve been saying in the talks I give about this research that doesn’t appear strongly in the paper is that I’m not quite satisfied with the Scapino classification system as it applies to archosaurs and other reptiles. This system (Class I-IV) generally describes the overall morphology of the symphyseal plate–the ligamentous surface of the dentary that attaches to the other dentary. Using it was a good start since numerous mammal-centric papers use it to describe the joint. And I think it does an adequate job of broadly categorizing chins into unfused vs fused joints. But this does little to tell us about the internal architecture of fibers (which vary among animals, and may be woven, parallel, and/or distributed differently in the joint), the role Meckel’s cartilage has in the joint, good ole variation, and other features of the chin such as integument, dentition, the predentary, and even its complementing premaxilla. My brain shuts down a bit when I think about how to study not only the symphysis, which is complicated enough, but the symphysis+premax as a single functional unit. ow. So, we’re digging more deeply into the dark corners of characters to better describe all of this beautiful, functionally insightful anatomy (See Protosuchus figure) as well as more quantitatively explore the evolutionary and developmental patterns underpinning the joint over time.

Gorgeous MicroCT data of Protosuchus (MCZ 6727) and its sexy chin.

Gorgeous MicroCT data of Protosuchus (MCZ 6727) and its sexy chin.

Stay tuned for more symphyseal goodness in the near future. As for access to the paper, for now, email me if you want a copy.

Citation: Holliday CM, Nesbitt SJ. 2013. Morphology and diversity of the mandibular symphysis of archosauriforms. Geological Society, London, Special Publications v379. 17pp. doi 10.1144/SP379.2.

Abstract: Archosauromorphs radiated into numerous trophic niches during the Mesozoic, many of which were accommodated by particular suites of cranial adaptations and feeding behaviors. The mandibular symphysis, the joint linking the mandibles, is a poorly understood craniomandibular joint which may offer significant insight into skull function and feeding ecology. Using comparative data from extant amniotes, we investigated the skeletal anatomy and osteological correlates of relevant soft tissues in a survey of archosauromorph mandibular symphyses. Characters were identified and their evolution was mapped using a current phylogeny of archosauriforms with the addition of non-archosauriform archosauromorphs. Extinct taxa with the simple Class I condition (e.g., proterochampsids,rauisuchians”), rugose Class II (aetosaurs, protosuchians, silesaurids), and interdigitating Class III symphyses (e.g., phytosaurs, crocodyliforms) and finally fused Class IV (avians) build the joints in expected ways, though they differ in contributions of bony elements and Meckel’s cartilage. Optimization of the different classes of symphyses across a archosauromorph clades indicate that major iterative transitions from plesiomorphic Class I to derived, rigid Class II-IV symphyses occurred along the lines to phytosaurs, aetosaurs, a subset of poposauroids, crocodyliformes, pterosaurs, and birds. These transitions in symphyseal morphology also appear to track with changes in dentition and potentially diet.

Transactions of the Royal Sounds of SVP

Yes, the seats squeaked upon sitting on them at this year’s Society of Vertebrate Paleontology meeting. During talks, most people sought to avoid the chirping by gently sliding laterally onto the cushion. But at the banquet, every applause was followed by waves of seat barks. Stay classy SVP!

All in all, the meeting was well executed, at a great location. Well done Host and Program Committees, and all the participants that made it possible.

Travels to Rockefeller State Refuge

Alligator hanging out along Route 82

Rockefeller State Refuge is an expansive area of the western end of Louisiana’s swampy coast which prides itself as being one of the key DNR sites to aid in the rescue of American Alligators when they were endangered several decades ago (Link to Map and Location).  Today, besides maintaining a large wildlife management area full of birds, fish, herps, and sporting enthusiasts, they manage the region’s alligator population, work with commercial farmers and supply most of the alligators used in research in North America. If you didn’t already know, research in alligators is booming. There is strong interest in alligator and crocodilian genomics, hematology and disease resistance, biomechanics (for example…the death roll..), cardiopulmonary and developmental physiology, let alone our persistence in using them as a comparative anatomical model for vertebrate paleontology, functional anatomy and evolution.

Early morning Sun, Rockefeller Jan 31, 2011

 In February 2011, Henry Tsai and I drove down to collect alligator cadavers and made it back to Columbia at 3am, about 1hour before a blizzard hit and snowed us in for 2 days. It was a drive of legend in which we threaded the needle between two separate winter storms driving up through Western Arkansas. The alligators were quite comfy in the back of the truck, in my driveway while  we were snowed in.

The morning after picking up gators, after a 15hr drive through winter weather, Feb 1, 2011.

Needless to say, there are few contrasts in weather than experiencing sunrise over a balmy swamp one morning, and then 24 inches of snow the next.  The gators were used for research as well as a fairly popular high school workshop “Inside Alligators”we put on a week or so after we returned.

This June, I was accompanied by Ohio U/Witmer lab alum and current Mizzou Lecturer Dave Dufeau, and two undergraduates, Cortaiga Gant and Julie Tea. Cortaiga has been part of Project Gator Chin for a while whereas Julie is a Visiting Summer Fellow from University of Houston. This trip made for a good experience for them–none of whom had ever visited a place like Rockefeller and everyone got a chance to see, hold, and work with alligators. We stopped in Baton Rouge on the way down, stuffed our faces with shrimp and oysters at The Chimes and had a relatively leisurely drive down the coast the next day to get to Rockefeller. We then drove the entire way back to make sure the eggs and cadavers were taken care of.

Beach West of Cameron, LA

We visited Rockefeller this trip to load up a bunch of eggs we are currently incubating in the lab. Alligator eggs typically take about 65 days to hatch and are laid in a large, mother-guarded, mounded nest in late May/Early June. The Refuge staff use helicopters and airboats to identify and flag nests every season. They also collect recently-laid eggs and keep them in large outdoor incubators. The eggs are given to researchers or are hatched and raised until they are released into the wild or used otherwise.

Julie, Dave, and Cortaiga at Rockefeller, June 2012

Rockefeller has something different going on every time I visit. I’ve seen other students there collecting blood and gut bacteria. This time they had a number of alligator gar carcasses being prepped for someone’s study. Rockefeller received some serious damage from Hurricanes Rita and Katrina in 2005. One the “major” losses was their large outdoor, walk-in freezer, which had dozens of frozen specimens including the head of a gator that must have been pushing 10feet long…all swept away. None of this would be possible without the support and effort of Supervisor Ruth Elsey. Ruth has championed research and made the resources of the Refuge available to researchers and students from around the world. Ruth is always keen to help out people with research projects and educational materials and is always a welcoming host at Rockefeller.

Holding Gators

Alligator gar heads

Ruth showing Julie and Cortaiga a local gator nest. Momma is just under the reeds at the bottom of photo.

Cartilage Fusion in Gator Chins

Mandibular joints and Meckel’s cartilage in Alligator.

Summer in the Holliday Lab is getting exciting. A busy Spring has resulted in a couple new projects coming out later in the Fall (more later) but we’re deep in new directions in the lab including our first stint into Evo-Devo. Some students and I traveled to Louisiana to retrieve about 100 Alligator eggs to run an experiment this summer on the development of the Alligator chin. We’re keen to understand when and how the chin, or mandibular symphysis develops and pinpoint particular mechanisms and events that occur during its in ovo transformation.

Meckel’s cartilage is the cartilaginous rod that provides the scaffold for the ossifying mandible and 1st Branchial arch derivatives in the vertebrate skull. So, in reptiles, it promotes the ossification of the bones of the lower jaw and quadrate and remains as a persistent cartilage throughout life (Top Figure). This is in stark contrast to the mammalian condition in which the cartilage cavitates, aids in the formation of the dentary, 2 middle ear ossicles, and the tympanic ring. This caudal end of the system is better known-with questions involving the evolution and development of the mammalian ear driving many research directions. If you want to read more, I point you to the research of Abigail Tucker and  Zhe-Xi Luo, among many others.

Cross-section through suture and Meckel’s cartilage and CT-based 3D model of mandibular symphysis with bone, and without showing Meckel’s cartilage and sutural ligament.

At the rostral end of Meckel’s cartilage, out at the mandibular symphysis, we find animals doing a couple of different things. More often than not, the two cartilage rods (Left and Right) remain independent of one another and the symphysis is solely a membranous, ligamentous syndesmodial joint that fully fuses in primates, oviraptors, neoavians and a handful of other vertebrates, or may be sutured but unfused (dogs, crocodyliforms) (Middle Fig), or even mobile as in snakes, some lizards, possums and other animals probably. Second, research into the development of the human chin has found that the two cartilaginous rods stick together during development and eventually recede, occasionally leaving small nodular cartilages. Although the 2 cartilages stick to one another, the perichondral layer remains patent. We found a similar pattern in Iguana chins in our 2010 Anat Rec paper on lizard mandibular symphyses. Third, and now to the point. In alligators and geckos, Meckel’s cartilage not only sticks to its opposite member, but it obliterates the perichondral borders between the two cartilaginous rods, forming a continuous cartilaginous rod from the left articular all the way around the chin and back to the right articular.

That’s cool! Buy why? Is it adaptive? We’ve got some ideas. How? we’re working on it. Why do we care? working on that too :) but in general, mandibular symphyses are important cranial joints functionally, so understanding their development and evolution are key goals if you want to understand the how the vertebrate head works. Meckel’s cartilage is often entwined in craniofacial defects that affect branchial arch development. Archosaur chins are really cool and quite diverse and thus may shed light on form, function, and ecology of feeding during their evolution.  Also, paleontologists have started to wiggle chin joints with more frequency these days, what with various animation and modeling applications,  despite there being what I consider a total lack of published research on how the joint is built (my fault I guess) and functions (in the works!)  in living reptiles.

Cleared and Double-stained alligator embryo showing the incipient double fusion and spatulate process of Meckel’s cartilage at the symphysis.

I digress… Fourth…Alligators take this cartilage fusion one step further in that a spatulate structure extends rostrally from the main body of Meckel’s cartilage  and forms a second fusion which eventually seals up with the caudal, main body of the cartilage. This spatulate form persists throughout life in alligators, leaving a large, flat trough within the bony symphysis. Is this fusion similar to those fusions we see in the hyoid skeleton, or the chondrocranium? probably. We’ll find out.

This summer we’re growing up Alligators to capture the time during development when this fusion occurs–I’ll post about that next.

Alligator Sesamoid Anatomy

We are happy to present a new project authored by Henry P. Tsai and myself entitled “Ontogeny of the Alligator Cartilago Transiliens and Its Significance for Sauropsid Jaw Muscle Evolution” which is out in PLoS ONE this week. The link to the paper is here. 

The paper describes a nodular structure characteristic to crocodilian jaw muscles known as the cartilago transiliens. Despite its familiarity to morphologists, anatomists, and the like, few studies have focused much on it. We tested the hypothesis that the structure is actually a sesamoid, or an intramuscular nodule, linking two historically disparate muscles. Historically, its been treated as a special structure without any particular developmental history. By using imaging, dissection and histology methods on a sample of different-aged alligators, we found that, based on a number of criteria, indeed the cartilaginous nodule is likely a sesamoid. Not earth-shattering research but it holds significance for understanding how the jaw muscles function, how they develop,  how they evolved among reptiles and the nature of the pterygoid buttress system of crocodilians. I had stumbled on this idea about the sesamoid/cartilago transiliens back when we published the 2007 J Morph Archosaur Jaw Muscle Homology paper. So, it’s good to see another paper spawn out of that work.

Besides integrating classical dissection and histology, the project was fun because we got to employ a relatively new staining technique using Lugol’s Iodine (I2KI) and MicroCT to visualize the 3D anatomy of the jaw musculature. I hinted at some of this last year and it’s good to see our first shot make it to press. Its remarkable how different iodine-enhanced CT is compared to MRI, which is sometimes used for muscle anatomy…though it’s best for brain and nervous tissue.

Similar coronal sections through same Alligator specimen using MicroMRI (Left) and Iodine-enhance MicroCT (Right)

We have put together a 3D model and pdf of the dataset, which is featured extensively in the paper. We bumped into a couple of technical difficulties at the last minute this week, so we aren’t able to launch at the same time the paper comes out (darn) however, it’ll be up soon enough. We’ll be employing this technique often in the Holliday Lab as it’s proven essential to understanding the 3D anatomy of muscles and other soft tissues that were always a challenge to convey on 2D figures, and almost impossible to get at using standard CT scanning and even MRI. Had this technique been around during my dissertation, it would’ve been a completely different monster. So jaw muscles v2.0 here we come.

The real milestone here is that this paper marks the 1st paper published with my 1st graduate student. hoo-rah. Henry is keen on limb anatomy moreso than heads, but in our Integrative Anatomy program, we have graduate students perform research rotations within their advisor’s lab as well as in other neighboring labs in IA or in other departments. This let’s them learn several subjects and techniques while they develop ideas for dissertations, get to know labs, etc. We thought this project would be a great compromise on learning bread & butter techniques in the lab, connective tissue biology & cartilage while working on a small soft tissue anatomy project on an animal we’re both quite fond of. Henry rocked it and was key in seeing its publication basically one year after he started graduate school.